Skin aging is a multifactorial process driven by both intrinsic factors (e.g., effects of natural course of chronological aging) and extrinsic factors (e.g., environmental toxins/pollutants and smoking).
The skin comprises, in particular, keratinocytes, melanocytes and nerve cells, fibroblasts, Langerhans cells, endothelial cells, smooth muscle cells, etc. Referring to FIG. 1, the keratinocytes and melanocytes are present in the epidermis. The melanocytes are located in the basal layer of the epidermis, and they constitute the site for melanogenesis. Due to their close contact with the keratinocytes, the melanocytes transfer to the keratinocytes the newly synthesized melanin in the form of melanosomes, thereby giving the skin its coloration. While melanocytes actually produce the pigment response, keratinocytes produce epinephrine, a catecholamine which is reported to trigger the melanocyte to produce pigment. This triggering of melanin via catecholamines, such as epinephrine, is called catecholamine biosynthesis, which will be described below. Thus, these two cell types work together symbiotically to modulate pigmentation. The type and the amount of melanin distributed in the keratinocytes determine the primary coloration of the skin.
Referring to FIG. 2, melanogenesis, or melanin synthesis, is a biological phenomenon initiated by hydroxylation of the L-tyrosine amino acid resulting from the formation of L-dihydroxyphenyl-alanine (L-DOPA), which is in turn converted to DOPA-chrome by the action of a specific melanocyte-associated enzyme, tyrosinase. Consecutive reduction and oxidation reactions result in the conversion of the DOPA-chrome to melanin. The production of tyrosinase and its activity determine in part the amount of melanin produced. The amount and the type of melanin transferred to the keratinocytes determine, for their part, the degree of visual pigmentation of human skin.
Skin aging or the appearance of skin aging is a function of melanogenesis. Skin aging or age spots are perceived as a contrast in skin tone. As used herein, “age spot” generally refers to spots on the skin associated with aging, and may encompass solar lentigo, freckles, melasma, seborrheic keratoses, post inflammatory hyperpigmentation, etc. As used herein, “tone” generally refers to the visual perception of a smooth and evenly-pigmented surface. Skin aging affects texture and pigmentation, creating contrasts on the face that may be a result of shadows caused by wrinkles or color changes caused by age spots. In young skin, melanin is evenly distributed, and melanocyte activity is low, restricted to the production of constitutive pigmentation only. UV radiation in sunlight transiently activates melanocytes to produce melanin that is evenly distributed, as in a tan. In aging skin, some melanocytes may be damaged by cumulative UV exposure, causing them to be permanently “switched on” and overproduce melanin. This overzealous melanogenesis production can eventually create permanent local discoloration with sufficient size and contrast to appear as age spots (lentigines) or as diffuse hyperpigmentation. As skin turnover decreases with age, microscopic bits of melanin (“melanin dust”) can become trapped in the epidermis and stratum corneum, contributing to a duller appearance. Uneven distribution of melanin manifests itself as age spots and hyperpigmentation. Hyperpigmentation in the age spot tissue relates to one or more of the following known factors: higher activity of tyrosinase, an above described melanocyte enzyme, and a higher instance of matured melanosomes and a higher retention rate of melanin/melanosomes in melanocytes or keratinocytes after they are synthesized.
Generally, UV radiation induced hyperpigmentation is thought to partially contribute to the actions of propiomelanocortin-derived peptides and a melanocyte stimulating hormone on the melanocyte melanocortin-1 receptor by increasing intracellular cAMP. Studies in propiomelanocortin-deficient mice and in animals with nonfunctional melanocortin-1 receptors reveals that they are still able to produce melanin in response to forskolin suggesting that alternate cAMP dependent pathways also induce melanogenesis.
One such alternate cAMP-dependent pathway involves the adrenergic receptors, which are pharmacologically divided into two groups: α and β. The adrenergic receptors are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. There are 3 types of beta adrenergic receptors: ADRβ1, ADRβ2 and ADRβ3, subtyped on the basis of differential pharmacological response to catecholamines and specific antagonists as well as differences in protein sequence. Moreover, there are multiple subtypes of alpha adrenergic receptors: ADRα1A, ADRα1B, ADRα1D, ADRα2A, ADRα2B, and ADRα2C.
According to Schallreuter, K. U. et al., The induction of the α-1-adrenoreceptor signal transduction system on human melanocytes, Experimental Dermatology 1996; Vol. 5, Issue 1, pages 20-23, human melanocytes do not express α1, β1 or β2 adrenoreceptors without extracellular stimulation. Introduction of norepinephrine causes a time-dependent induction of α1 receptors; however, there is no effect on melanogenesis. In contrast β-adrenergic receptors in melanocytes were not induced by norepinephrine stimulation, thus norepinephrine synthesized by the melanocyte does not appear to mediate pigmentation/melanogenesis.
In contrast, induction of catecholamine biosynthesis in keratinocytes correlates with an increase in ADRβ2 receptors. Schallreuter conducted studies which implicated ADRβ2 signaling pathways and catecholamine synthetic networks within the epidermis. Both keratinocytes and melanocytes express ADRβ2 and both cell types have the enzymatic machinery for catecholamine biosynthesis, specifically norepinephrine synthesis, although only keratinocytes are known to synthesize epinephrine. The secretion of epinephrine from the keratinocytes stimulates the beta adrenergic receptors on the melanocytes, which increases intracellular cAMP, and thereby increases melanogenesis.
Sivamani et al., An Epinephrine-Dependent Mechanism for the Control of UV-Induced Pigmentation, Journal of Investigative Dermatology (2009), vol. 129, pages 784-786 disclosed that keratinocytes secrete epinephrine in response to UV radiation, and the epinephrine stimulates the beta adrenergic receptors on melanocytes to increase melanin synthesis. Thus, Sivamani established a link between stress, UV radiation, and pigmentation. Additionally, Sivamani confirmed the general consensus also provided in Schallreuter that α1 receptors have no effect on melanogenesis.
Grando, Adrenergic and Cholinergic Control in the Biology of Epidermis: Physiological and Clinical Significance, Journal of Investigative Dermatology Vol. 126, pages 1948-1965 (2006) teaches the epidermal adrenergic signal controls calcium homeostasis, and suggests that pigmentation may be controlled via the β2 and α1 adrenergic receptors; however, Grando's examples centered on the relationship of ADRβ2 to melanogenesis. Grando further asserts that melanocytes express only ADRβ2 receptors, whereas ADRβ1 receptors are considered absent.
A greater understanding of the biochemical processes responsible for aging, such as the mechanism described above, has invigorated the cosmetics industry and resulted in the emergence of a new class of cosmetic actives sometimes referred to as “cosmeceuticals.” The purported effects, including but not limited to antioxidant, anti-inflammatory and free-radical-scavenging effects, derive from the underpinning science.
The present inventors recognized the deficiencies associated with evaluation of known cosmetic agents for cosmetically functional effects and the deficiencies associated with the identification of agents that actually achieve a theoretically-based effect to provide actual, verifiable anti-aging benefit to skin. Accordingly, the present inventors recognized a continual need for reliable and efficient in-vitro methods to identify agents effective in reducing the size and intensity of age-related skin spots, specifically by modulating the production of melanin. While the literature has established a link between ADRβ2 receptors and melanogenesis generally, the linkage between adrenergic receptors and melanogenesis in age spots is not well established, and there is still a need to identify additional adrenergic receptors which modulate melanogenesis in age spots.